Multidrug-resistant extended spectrum β-lactamase (ESBL)-producing Escherichia coli from farm produce and agricultural environments in Edo State, Nigeria

Antimicrobial resistance (AMR) is a major public health concern, especially the extended-spectrum β-lactamase-producing (ESBL) Escherichia coli bacteria are emerging as a global human health hazard. This study characterized extended-spectrum β-lactamase Escherichia coli (ESBL-E. coli) isolates from farm sources and open markets in Edo State, Nigeria. A total of 254 samples were obtained in Edo State and included representatives from agricultural farms (soil, manure, irrigation water) and vegetables from open markets, which included ready-to-eat (RTE) salads and vegetables which could potentially be consumed uncooked. Samples were culturally tested for the ESBL phenotype using ESBL selective media, and isolates were further identified and characterized via polymerase chain reaction (PCR) for β-lactamase and other antibiotic resistance determinants. ESBL E. coli strains isolated from agricultural farms included 68% (17/25) from the soil, 84% (21/25) from manure and 28% (7/25) from irrigation water and 24.4% (19/78) from vegetables. ESBL E. coli were also isolated from RTE salads at 20% (12/60) and vegetables obtained from vendors and open markets at 36.6% (15/41). A total of 64 E. coli isolates were identified using PCR. Upon further characterization, 85.9% (55/64) of the isolates were resistant to ≥ 3 and ≤ 7 antimicrobial classes, which allows for characterizing these as being multidrug-resistant. The MDR isolates from this study harboured ≥1 and ≤5 AMR determinants. The MDR isolates also harboured ≥1 and ≤3 beta-lactamase genes. Findings from this study showed that fresh vegetables and salads could be contaminated with ESBL-E. coli, particularly fresh produce from farms that use untreated water for irrigation. Appropriate measures, including improving irrigation water quality and agricultural practices, need to be implemented, and global regulatory guiding principles are crucial to ensure public health and consumer safety.


Introduction
The usage of antimicrobial drugs in veterinary and human medicine has led to the extensive development of antimicrobial resistance (AMR) in animals, humans, and the environment [1][2][3]. A significant concern is how AMR disseminates rapidly globally [4,5]. Third-generation cephalosporins are essential in our defence against bacterial infections [6]. Exposure to 3 rd generation cephalosporin-resistant Escherichia coli (E. coli) may occur through different routes, such as interaction with animal or human carriers, consuming contaminated food or interaction with contaminated environmental compartments [7]. It is pivotal to elucidate the relative involvement of probable routes and sources to institute preventive mechanisms.
Various market segments, from consumers to restaurants and institutional markets, are regularly supplied with fresh produce in Nigeria. An enormous quantity of ready-to-eat (RTE) vegetables is retailed, particularly street foods [8]. Several outbreaks have highlighted fresh vegetables as potential vehicles of foodborne disease [9]. Although not in outbreak proportions, E. coli illnesses have also been reported in Southern Nigeria [10][11][12]. Its heightened concern over the spread of antimicrobial-resistant pathogens made this research imperative. Nigeria currently does not have a functional foodborne disease reconnaissance system. Its direct healthcare consequence is projected at approximately $3 billion, responsible for 17-25% of the projected cost for all illnesses [13]. Nigeria's Area Councils Health System profile showed foodborne infection (FBI) as the foremost cause of mortality, averaging 25%. In Nigeria, >200,000 people die annually from food poisoning caused by foodborne pathogens, as estimated by the World Health Organization (WHO) [2,8,14]. Mortalities result from contaminated foods via improper preservation, service and processing [15].
Fecal E. coli can occur in the soil, water and on plants. ESBL-producing pathotypes of E. coli have also been recovered in the environment [16][17][18]. Since vegetables are usually eaten raw, ingesting ESBL-producing E. coli that can reach the gastrointestinal tract poses a potential public health risk [6,14,19]. In recent decades, the prevalence of multidrug resistance (MDR) E. coli has been increasing globally [20,21]. Selective pressure from antibiotics, which culminates from their usage and abuse, contributes to AMR [3,22]. A key mechanism for resistance to third-generation cephalosporins is centred on the plasmid-mediated enzyme (β-lactamase) production, which inactivates these compounds by cleaving the β-lactam ring. CTX-M is one of the most predominantly documented ESBL β-lactamase types from clinical environments in E. coli isolates [23].
ESBL-producing E. coli from animals and humans can enter the environment through manure and sewage. Fresh produce may become contaminated with ESBL-producing E. coli from the farm environment by irrigation with contaminated water sources or when cultivated in manure-supplemented soil. ESBL-producing E. coli could persist on vegetable surfaces and sometimes advance to the interior and spread to animals or humans [24,25]. In recent years, interest has focused on the microbiological hazards that challenge the food industry [1,8,14]. It is imperative to pinpoint the crucial role of consuming uncooked or undercooked vegetables in bacterial gastroenteritis [26]. There are limited studies on spreading and transmitting ESBLproducing E. coli in agricultural environments and food vegetables [7,27]. The incidence of ESBL E. coli in food, vegetables, and the farm environment is detrimental to public health and food safety. The current study intended to evaluate the presence of ESBL-producing E. coli on food vegetable samples in Edo State, Nigeria. Ready-to-eat (RTE) salads and vegetables that are potentially consumed uncooked were selected for isolation and characterization of ESBL E. coli isolates, as were vegetables from farms and markets. Vegetables from farms were included to reflect the occurrence of ESBL-producing E. coli in the agricultural environment.

Study area and sample collection
The sample size for this study was determined using the sample size determination formula below: Z 1-α/2 = Standard normal variant at 5% type I error (P < 0.05); P = Expected prevalence of 4.0-10.12% was based on previous studies [7,21,[28][29][30][31][32][33]; d = Absolute error or precision (which is 5%). The expected minimum sample to be collected ranged from 59-140. Edo State was selected to pilot the study due to its strategic location in Southern Nigeria and its largescale cultivation of vegetables due to its conducive climate and environmental conditions. There are eighteen Local Government Areas (LGA) in Edo State, and samples were collected in each LGA (Figs 1 and 2). A total of 254 samples were examined. Edo South is the most populated among the three senatorial districts and is also where the state capital (Benin City) is located (Fig 1).
LGA are geographical entities within a state in Nigeria which largely depend on the land mass of the states, and Local Government Councils administer these. The senatorial districts in Nigeria consist of a combination of LGA within a state, with each state consisting of three senatorial districts. A single representative farm was surveyed from each LGA, Samples from agricultural farms include soil

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(n = 25), manure (n = 25), irrigation water (n = 25), and vegetables (n = 78). Samples from vendors and open markets include ready-to-eat (RTE) salads (n = 60) and vegetables potentially consumed uncooked (n = 41). The fresh produce from vendors and open markets potentially consumed uncooked included: lettuce (n = 7), carrot (n = 7), tomato (n = 7), cabbage (n = 6), pepper (n = 7) and cucumber (n = 7). Ethical approval was not obtained before sampling collection since samples were not obtained directly from humans or food-producing animals. However, verbal informed consent and permission were obtained from the farm owners before sampling collection from respective farms.

Data analysis
The data from this study were analyzed using Microsoft Excel 2013 and SPSS 21.0. The prevalence and occurrence from this study were expressed in percentage (%) while the population density from the samples was expressed as mean ± standard deviation of the mean.

Prevalence of ESBL E. coli from agricultural farms and open markets
A total of 64 E. coli isolates were identified using PCR and further characterized ( A total of 85.9% (55/64) isolates were resistant to �3 and �7 antimicrobial classes, which categorizes these as being multidrug-resistant (MDR). The multiple antibiotic resistance (MAR) index of the isolates ranged from 0.27 -to 0.62 for these 55 MDR isolates. However, all the isolates were multi-resistant (resistant to a minimum of 4 antibiotics) and displayed 52 resistance phenotypes (Table 3). A MAR index >0.5 was observed to be associated with isolates recovered from the agricultural environments. In addition, isolates from agricultural environments seemingly showed a higher resistance phenotype than isolates from vendors and open markets. The phenotypic resistance phenotype of the isolates was independent of the beta-lactamase determinants of the isolates ( Table 3).

Presence of resistance determinants in ESBL E. coli isolates
The proportions of ESBL E. coli isolates that harboured beta-lactamase genes in 3.1% (2/64), intI1 20.3% (13/64), intI2 3.1% (2/64). The 55 MDR isolates harboured a minimum of 1 and a maximum of 5 AMR determinants. The 55 MDR isolates also harboured a minimum of 1 and a maximum of 3 beta-lactamase determinants. The detection of the betalactamase determinants of the isolates was independent of the detection of other antibiotic resistance genes. All isolates harbouring respective antibiotic resistance genes showed phenotypic resistance to �1 antibiotic from the antimicrobial class (Table 3).

Discussion
Vegetables are highly prone to contamination by microorganisms through contact with soil, water and handling during harvest or after-harvest processing. They may therefore harbour a diverse range of bacterial pathotypes, including human and plant pathogens. Quarcoo et al. [21] reported that counts expressed in CFU/g ranged from 186 to 3000, with the highest counts found in lettuce, with all lettuce samples positive for E. coli. Though the population density from Quarcoo et al. [21] study is similar to ours, the occurrence rate is higher than ours. This could be attributed to the selective characteristics of the media used in our research. Reported microbial load range of <3 to >1100 MPN/g from Vegetables in Malaysia [41] and 2.31 ±0.86 log CFU/g to 5.50 ±0.7 log CFU/g in Spain [31] were also similar to those of our study. The total counts of ESBL-E. coli from the farm produce and environments were determined to evaluate their microbiological quality and safety. The potential sources of ESBL-E. coli contamination could result from faecal contamination, poor water quality, poor distribution and sanitary conditions, and poor handling practices. Lower ESBL-E. coli prevalence from farmed vegetables compared to those of our study have been reported from Finland, Spain, Germany,        [47] documented that 25.7% of retail store samples determined were positive for ESBL isolates, which was more comparable to the results of this study. Soré et al. [48] reported that 42.47% of salad samples were positive for ESBL-producing isolates. On the other hand, that prevalence was higher than the prevalence recovered from our study, and the differences may be explained by different soils, manure and agricultural practices of the other regions/countries. Variations in the prevalence of ESBL-producing E. coli isolates depend on the sample size and types of vegetables tested. ESBL strains have been occasionally found in human infections and foods [8,49,50]. In a recent populationbased modelling study, 18.9% of human ESBL-E. coli carriage was attributed to a food source [51], highlighting the importance of AMR surveillance and hygiene measures for food products.
In this study, high heterogeneity in the presence of ESBL E. coli was observed both at the farm level and from open markets. Njage and Buys [52] documented an increased incidence of ESBL-producing E. coli in irrigation water (73%-64%) samples and lettuce at harvest (90%) in South Africa. A higher prevalence was recounted from the irrigation water in this study. In the Netherlands, irrigation water (100%) and harvested lettuce (14.7%) were also found to harbour beta-lactamase-producing Enterobacteriaceae [7]. The incidence in irrigation water was far higher than observed in this study. Using a discrete-time model by Costa et al. [53], it was reported that ESBL-E. coli prevalence reached an equilibrium prevalence of 0.65% in the open community, where the colonization of the open community could primarily be attributed to the open community itself (62%), followed by vegetable consumption (29.5%), and contact with farmers (8.5%). A clear link between irrigation water and leafy green vegetable E. coli isolates has been established [54]. As low-income countries have higher AMR abundance in their

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wastewater [20,22,55,56], the risk of ESBL dissemination is greater. Using contaminated irrigation water may directly link to AMR contamination, mainly if the water is applied to edible parts of fruits and vegetables [57]. The types of nutrients that are frequently present on the leaf surface of leafy green vegetables can be categorized into two: inorganic (ions) and organic molecules (organic acids and carbohydrates) [58]. Carbohydrates (dominant phyllosphere sugars are glucose, fructose and sucrose) are of particular interest due to their capacity to readily support the growth of enteric bacteria such as E. coli. Different factors could again account for these differences, but using untreated wastewater, seasons and manure application as fertilizers might be significant factors [58]. Some authors have previously documented the identification of ESBL E. coli and the possible transmission of antibiotic-resistant bacteria via treated wastewater for irrigation [58,59], while others could not find a clear connection [60].
Concerns about the quality and safety of vegetable salads and fresh produce have increased, despite their essential nutritional benefits to humans [6][7][8]14,19]. Outbreaks associated with fresh produce portray a dearth of information crucial in understanding the ecology of ESBL E. coli outside animal and human-animal hosts [61]. The microbial incidences reported for fresh produce could be attributed to contaminated cultivated soil, poor storage methods and poor agricultural practices by handlers. The increased microbial occurrence on leafy greens may be linked to the structure of their surface, with many folds and furrows and a corresponding high surface area, which may lend itself to colonization. Fresh produce contamination by pathogenic bacteria is currently emerging as a significant food safety concern, and the occurrence of MDR bacteria can signify an additional hazard. Prevalence of MDR (96.1%) and resistance to aminoglycosides (46.7-66.7) in ESBL-producing Enterobacteriaceae have been reported previously [47,62], which is similar to the findings from this study. Richter et al. [63] reported that 98% of their isolates showed an MDR phenotype, close to the 100% of our research. Vegetables are often eaten without heating processes, leading to a greater risk of acquiring resistant bacteria [6,14,19]. Microbial food quality control maintains a critical factor in continually controlling foodborne illnesses (one of the primary causes of mortality and morbidity) coupled with a cost-intensive loss for food companies.
Contamination by ESBL E. coli of farm environments and vegetable samples from our study hypothetically could primarily be from animal sources. Since the manure used in farm environments is predominantly from animal sources. These animals are usually treated with antibiotics for therapeutic and prophylactic purposes [64,65]. These antibiotic residues could be excreted by the animal in an unmetabolized or poorly metabolized form, resulting in the development and transmission of AMR [66]. But also, harvesting and handling by workers that harbour the pathogens may be a contamination source; thus, cross-contamination is another factor that needs to be considered in spreading antimicrobial-resistant bacteria from foods to humans [67]. In addition, the potential implication of untreated surface water or wastewater for irrigation purposes in Edo State, Nigeria, cannot be overemphasized. An increase in AMR gene carriage in soil has been documented for years [68], pinpointing the role of soil as a reservoir for genes and antibiotic-resistant pathogens. Given that the soil community could harbour these genes and AMR bacteria, contamination during postharvest may also arise from this source in RTE salads and fresh vegetables. This could be an essential route for the spread of antibiotic-resistant pathogens.
Soré et al. [48] documented that resistance to the aminoglycoside, fluoroquinolones and sulfonamides classes was dominant in fresh-cut salads and wastewater samples. An average of 50% of ESBL-E. coli isolates from our study were resistant to aminoglycoside and fluoroquinolones, with 35.9% resistant to the sulfonamides class of antimicrobials. Zurfluh et al. [47] reported that all isolates from the investigated fresh vegetables were resistant to cephalothin and ampicillin, while 53 (88.3%) were resistant to cefotaxime. All our isolates were resistant to cefotaxime and ceftazidime, with additional resistance to cefpodoxime 96.9% (62/64), ceftriaxone 98.4% (63/64) and ampicillin 48.4% (31/64). Among 12 antibiotics screened by Ramadan et al. [69], only imipenem seems to have remained resilient, which was in line with the carbapenems activity from our study. The high resistance to fluoroquinolones, penicillin, tetracyclines and aminoglycosides is probably a result of easy access, increased use and nonregulation of these antibiotics on the Nigerian market.
Manure from food animals, usually used as a soil fertilizer, may harbour different resistant bacteria and resistance encoding genes from the gut microbiota of food animals [65]. This phenomenon has become a major problem for public health, not only in underdeveloped countries but also in high-performing, socio-economically developed countries. ESBL E. coli was identified in vegetables from vendors of market origin in our study and has also been detected in the Netherlands [70] and Tunisia [27]. It is important to note that vegetables that harbour ESBL E. coli from open markets are usually consumed uncooked or undercooked, where the potential for transfer to humans cannot be ruled out [7]. Outbreaks of foodborne origin (vegetables) consequent of ESBL-E. coli isolates have been documented [71,72]. Above 50% of ESBL E. coli recovered could be disseminated via conjugation through ESBL determinants to other bacteria [27]. The horizontal spread of antibiotic-resistant determinants between soil and human bacteria has been reported previously [73]. The transfer of beta-lactamase elements in E. coli strains could be linked with IncI1 replicon plasmid acquisition [74].
Our results correlate with other studies indicating vegetables as a possible route for the dynamic diffusion of AMR genes in the community [58]. The bla ESBL and other resistance genes on vegetables have been described as existing predominantly in opportunistic and saprophytic bacteria, which are thought to represent a background reservoir of antibiotic resistance genes [22]. Iseppi et al. [75] reported that PCR confirmed the occurrence of bla  in the ESBL E. coli studied. Richter et al. [63] Genotypic characterisation showed the widespread prevalence of CTX-M-1, followed by SHV and TEM. CTX-M14 and CTX-M15 are widespread in the environment, food animals and humans in Europe [76,77]. bla TEM-1 and bla TEM-15 have also been reported from vegetables in Finland [44]. Similar β-lactamase genes to those from our study have been reported in Southern Thailand [45]. ESBL groups identified from our research (CTX-M-15 and CTX-M-1) also correspond to ESBL groups previously reported in Tunisia [27] from vegetables, irrigation water and soil; and in E. coli from healthy humans, food-producing animals, and food [74,78]. Hence, contamination by ESBL E. coli from farm environments and vegetable samples could have occurred from both animal and human origins in Nigeria. The probable consequence of untreated irrigation water can also not be overemphasized as contributing to the spread of MDR pathogens. CTX-M-15 ESBL was detected in our study and has been identified as the most common type of ESBL in Gram-negative bacteria worldwide from different sources, including humans, animals, wildlife and wastewater samples [58].
The finding of resistance genes (bla CTX-M-15 , bla CTX-M-1 , and bla TEM ) commonly linked to human sources [44] highlights the potential transmission route of AMR via food products and emphasizes the importance of hygiene measures. Zurfluh et al. [47] reported that 38.5% (10/ 26) isolates harboured bla CTX-M-15 , and 3.8% (1) were positive for bla CTX-M-1 . This is not in line with the findings from our study, as 10.9% (7) strains harboured bla CTX−M−15 , while 76.6% (49) harboured bla CTX−M−1. Ramadan et al. [69] reported that 90.4% (179/198) ESBL isolates contained �1 bla CTX-M gene, which was similar to the 85.9% (55/64) observed for our isolates. The prevalence of bla CTX-M-15 determinants in isolates has been documented in India following preceding studies from clinical isolates from South India and Delhi [79]. The group CTX-M-15 has progressively been described in community isolates and reported as the utmost Gram-negative ESBL bacteria in Europe [80]. Also, the CTX-M-15 group is especially prevalent in samples from the medical area in Germany [81], while the CTX-M-1 group more often occurs in samples from livestock [82]. The CTX-M-1 group was the most commonly found in this study (49/64; 76.6%, Table 3), which points toward a predominantly livestock origin of the ESBL isolates. Community-onset ESBL-associated infections, including urinary infection and sepsis, are principally caused by E. coli having bla CTX-M ESBLs [54]. Therefore, the relevant β-lactamase genes might contribute to the presence of ESBLs in community-transmitted infections from animal sources to humans via food chains.
The isolates in the present study carried AMR genes conferring resistance against critically essential antimicrobials [83], including aminoglycosides, third-and fourth-generation cephalosporins, tetracyclines and quinolones. All ESBL E. coli isolates from our study were MDR, and some carried integrons, as this characteristic is typical of ESBL-producing bacteria [84]. However, this was different from a survey by Soré et al. [48], where relatively less (93.23%) of the isolates showed MDR (94.40% in wastewater and 91.66% in salads). Co-expression of βlactamase genes may result in stronger ESBL production and increased antibiotic resistance or MDR [45]. Antibiotic resistance genes encoding 12 major classes of antibiotics, including βlactam antibiotics, aminoglycoside, macrolide, fluoroquinolone, and others, were found in vegetable samples from the USA, which were similar to the genes detected from samples from our study [46]. Compared to our findings, class 1 integrons were previously identified in MDR beta-lactamase-positive isolates [63,85]. Class II integron was not identified by Richter et al. [63], while it was identified at a low prevalence of 3.1% (2) in our study. Bezanson et al. [86] recounted that class 2 integrons were primarily identified in Canada, accompanied by class 1 integrons from salad vegetables. This is contrary to the findings from our study, where the class I integron was predominant. The diversity of resistance phenotypes observed among the ESBL-E. coli isolates showed that the potential spread of resistant bacteria is not the consequence of disseminating a unique strain. It instead appears that genetic variations of ESBLs aren't narrowly restricted or localized to a singular ecosystem. Farming practices, such as selecting organic fertilizers, types of irrigation water, and sanitation of vegetable processing environments, may affect the composition of the microbiome and antibiotic resistome in vegetables. Understanding the various potential pathways for antibiotic resistome dissemination through cultivation-irrigation-manure microbiomes is critical for mitigating antibiotic resistance. The limitations of this study are the fact that a single state from Nigeria was covered, and state-of-the-art molecular tools such as Whole Genome Sequencing (WGS), multilocus sequence typing (MLST) were not carried out to determine the sequence types (ST), and clones that are circulating with the farms and the market environments. However, the research group is currently working on attracting funding for such aspects of the research to monitor the microbiome, virulome and resistome using metagenomic tools.

Conclusion
Findings from this study represent the first investigation on the identification and characterization of ESBL E. coli recovered from farm environments, fresh produce and salads in Edo State, Nigeria. This work showed that fresh vegetables and salads could harbour ESBL-E. coli, particularly fresh produce from farms that use untreated water for irrigation. Antimicrobialresistant pathogens can spread to humans via the food chain, especially when these products are consumed uncooked. Suitable measures, for example, improvement of water quality and agricultural practices, need to be implemented, as well as established global regulatory guidelines to ensure public health and consumer safety. Close antimicrobial resistance surveillance in bacteria from a one-health perspective is crucial to source-track and mitigate the development and spread of such bacteria. Findings from this study could serve as baseline data to evaluate the human health risk and the potential transmission associated with the consumption of food vegetables. The subsequent aspect of this study includes expanding the survey to other zones in Nigeria, use of whole genome sequencing and multilocus sequence typing to characterize the isolates further to understand the exact clones that are circulating in the farm environments across Nigeria, Supporting information S1  45. Romyasamit C, Sornsenee P, Chimplee S, Yuwalaksanakun S, Wongprot D, Saengsuwan P. Prevalence and characterization of extended-spectrum β-lactamase-producing Escherichia coli and Klebsiella pneumoniae isolated from raw vegetables retailed in Southern Thailand. PeerJ 2021; 9:e11787.